Nucleotides Comprising CasPhi Modified for Nuclear Targeting

Abstract

The presently-disclosed subject matter relates to a compositions and methods that makes use of a CasΦ (CasPhi) that has been modified for effective nuclear targeting. In particular, certain embodiments of the presently-disclosed subject matter relate to unique nucleic acid molecules, compositions, and methods for delivery of CasPhi to the nucleus to effectively modulate expression or otherwise target of a gene of interest. A nucleic acid disclosed herein includes a nucleotide encoding a CasPhi, wherein there are no amino acid substitutions, or wherein proline residues at amino acids 749 and 753 have been substituted; a nucleotide encoding an amino-terminal linker connected to the amino-terminal end of the CasPhi; a nucleotide encoding a carboxy-terminal linker connected to the carboxy-terminal end of the CasPhi; and a nucleotide encoding a nuclear localization signal (NLS) downstream from the CasPhi and carboxy-terminal linker.

1 . A nucleic acid, comprising:
 a) a nucleotide encoding a CasPhi, wherein there are no amino acid substitutions, or wherein proline residues at amino acids 749 and 753 have been substituted;   b) a nucleotide encoding an amino-terminal linker connected to the amino-terminal end of the CasPhi;   c) a nucleotide encoding a carboxy-terminal linker connected to the carboxy-terminal end of the CasPhi; and   d) a nucleotide encoding a nuclear localization signal (NLS) downstream from the CasPhi and carboxy-terminal linker.   
     
     
         2 . The nucleic acid of  claim 1 , and further comprising a nucleotide encoding a protein domain for facilitating a CRISPR application. 
     
     
         3 . The nucleic acid of  claim 2 , wherein the application is selected from the group consisting of gene editing, imaging, transcriptional activation, and transcriptional repression. 
     
     
         4 . The nucleic acid of  claim 1 , and further comprising a repressor domain or an activator domain. 
     
     
         5 . The nucleic acid of  claim 1 , and further comprising a repressor domain downstream from the CasPhi and carboxy-terminal linker. 
     
     
         6 . The nucleic acid of  claim 5 , wherein the repressor domain is between the carboxy-terminal linker and the NLS. 
     
     
         7 . The nucleic acid of  claim 5 , wherein NLS is between the carboxy-terminal linker and the repressor domain. 
     
     
         8 . The nucleic acid of  claim 5 , wherein the repressor domain is selected from the group consisting of KRAB, SRDX, T1R1, MAD1, and TIEG1. 
     
     
         9 . The nucleic acid of  claim 8 , wherein the repressor domain is a KRAB-domain fusion. 
     
     
         10 . (canceled) 
     
     
         13 . The nucleic acid of  claim 5 , and further comprising a second repressor domain. 
     
     
         14 . The nucleic acid of  claim 13 , wherein the second repressor domain is connected to the amino-terminal end of the amino-terminal linker. 
     
     
         15 . The nucleic acid of  claim 1 , and further comprising a first promoter operably connected to the repressor domain or the dCasPhi; 
     
     
         16 . The nucleic acid of  claim 15 , wherein the first promoter is an RNA polymerase II promoter. 
     
     
         17 . The nucleic acid of  claim 15 , and further comprising a guide RNA (gRNA) and a second promoter operably connected to the gRNA. 
     
     
         18 . The nucleic acid of  claim 1 , and further comprising a guide RNA (gRNA) and a second promoter operably connected to the gRNA. 
     
     
         19 . The nucleic acid  claim 18 , wherein the second promoter is an RNA polymerase II promoter. 
     
     
         20 - 21 . (canceled) 
     
     
         22 . A composition comprising the nucleic acid of  claim 1  and a component for delivery. 
     
     
         23 - 25 . (canceled) 
     
     
         26 . A vector comprising the nucleic acid of  claim 1 . 
     
     
         27 . (canceled)